DNA binding protein

ABSTRACT

Methods of screening for compounds which are, for example, capable of modulating amino acid-DNA interaction, modulating DNA replication, modulating cell proliferation, and for identifying compounds which inhibit cellular proliferation caused by cancer, are provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/291,360 filed Dec. 1, 2005 which was a divisional of U.S.patent application Ser. No. 10/487,964 filed Feb. 26, 2004 which was a35 U.S.C. §371 of PCT Application No. PCT/US02/027809 which was filed onAug. 30, 2002 and claimed priority to U.S. patent application Ser. No.60/316,496 which was filed on Aug. 31, 2001.

FIELD OF THE INVENTION

This invention relates to methods of screening for compounds which are,for example, capable of modulating amino acid-DNA interaction,modulating DNA replication, modulating cell proliferation, and foridentifying compounds which inhibit cellular proliferation caused bycancer.

BACKGROUND OF THE INVENTION

The process of DNA replication is the primary target of anticancerchemotherapy, since cancer cells, like normal cells, have to replicatetheir DNA in order to divide. Identification of the proteins that areneeded for DNA replication can provide new candidates against which todesign novel anti-tumor drugs. To date, most anticancer drugs have beendirected against the enzymatic machinery of DNA synthesis. However, thegenetic mutations that lead to cancer are rarely found in these enzymes.Instead, it is the genes involved in signaling pathways controlling theinitiation of DNA synthesis during the cell cycle origin responding toerrors of replication that are most often mutated in cancer cells.

Chromosomal replication origins are molecular switches, where the majorregulated step of DNA synthesis, the initiation of replication, occurs.The applicants previously identified the origin of replication of thehuman c-myc gene. Characterization of the c-myc origin has led to thediscovery of a new protein, called DNA Unwinding Element binding protein(DUE-B) that binds in vivo in a yeast one-hybrid assay to an importantcontrol site in the c-myc origin, the DNA unwinding element (DUE).

Other relevant protein complexes are known in the art. For instance,U.S. Pat. No. 5,217,864 describes a replication initiator proteincomplex for eukaryotic cells that comprises a purified protein complexcapable of origin-specific DNA binding in vitro. While this protein hasnot been shown to function as such in vivo, it may be useful in thedevelopment of specific diagnostic and therapeutic applications, such asdrug assays to identify inhibitors of S phase initiation.

However, no protein has been identified to date with the property ofbinding in vivo to a region of DNA that controls DNA replication.Accordingly, the need remains in the art for new ways to interfere withthe cell division cycle.

It would therefore be desirable to identify a novel DNA binding proteinfor use as a target in pharmaceutical assays for chemotherapeutic drugsdesigned to inhibit tumor cell division, as well as to identify othercompounds effective in enhancing or retarding cell division.

It would also be desirable to prepare antibodies or antibody derivativesthat react with the DNA binding protein.

It would further be desirable to prepare cloned and purified forms ofthe DNA binding protein from bacterial and eukaryotic cells, as well asmodified forms of such a protein fused or not to other polypeptides.

SUMMARY OF THE INVENTION

The present invention meets these needs by providing a novel, purifiedhuman protein that binds in vivo to the c-myc replication origin DNAunwinding element.

In accordance with one aspect of the present invention, an isolatednucleic acid molecule is provided comprising a nucleotide sequenceencoding a human DNA binding protein, wherein the binding protein isDUE-B protein. The applicants have determined through other experimentsthat a certain region of DNA, the human chromosomal c-myc replicationorigin, regulates the initiation of DNA synthesis. The DUE-B protein hasbeen shown to bind in vivo to this region.

Additional features of the present invention include the preparation ofantibodies or antibody derivatives that react with the DUE-B protein,the preparation of cloned and purified DUE-B from bacterial andeukaryotic cells, and the identification of a frog (Xenopus laevis)protein related to human DUE-B. Still an additional feature of thepresent invention includes the preparation of modified forms of DUE-Bfused to other polypeptides.

DUE-B mRNA and protein are present constitutively through the cellcycle. DUE-B expressed in HeLa cells is localized in the nucleus, andcell fractionation experiments indicate that a portion (˜30-40%) ofintracellular DUE-B is bound to chromatin. Roughly 90-95% of theendogenous DUE-B protein extractable from chromatin in HeLa cellsassociates to form dimers that comigrate on gel filtration with DUE-Bexpressed from a baculovirus vector. The remaining 5-10% of DUE-Bextracted from HeLa cells migrates in a high molecular weight (>250 kDa)form. In contrast, human DUE-B expressed in bacteria chromatographs as26 kDa monomers. In vitro, human baculovirus expressed DUE-B binds DNAand interacts with the c-myc DUE in conjunction with other, as yetunidentified, proteins. The human DUE-B gene comprises seven exons onchromosome 20. Sequencing of the DUE-B cDNA suggested the presence of anATPase motif in the protein, and the ATPase activity has been confirmed.DUE-B is also a kinase substrate, consistent with the presence of sevencasein kinase consensus target sites in the protein. The DUE-B geneshows strong homology across evolutionary boundaries, from bacteria toyeast, mice, and humans. The carboxyl terminus of DUE-B displays aminoacid sequence homology to the human ERK5 kinase, and the human androgenreceptor, while the amino terminus of DUE-B shows strong homology to anevolutionarily highly conserved domain of unknown function (DUF154).

In Xenopus oocyte extracts, baculovirus expressed DUE-B proteinassociates to form high molecular weight complexes (>250 kDa) while theimmuno-crossreactive endogenous frog putative DUE-B protein migrates as˜50 kDa dimers. Similarly, in human cell extracts baculovirus expressedDUE-B protein associates to form high molecular weight complexes (>250kDa) while the endogenous human DUE-B protein migrates as ˜50 kDadimers. Baculovirus expressed human DUE-B promotes plasmid supercoilingand transiently inhibits DNA replication in these extracts. Restorationof DNA replication may parallel modification of the exogenous DUE-Bprotein.

Accordingly, it is a feature of the present invention to provide a novelprotein which can be used as a target in pharmaceutical assays forchemotherapeutic drugs designed to inhibit tumor cell division, as wellas to identify other compounds for enhancing/retarding cell division.This, and other features and advantages of the present invention, willbecome apparent from the following detailed description and accompanyingdrawings.

In accordance with the present invention, there is provided a nucleicacid sequence encoding a DNA binding protein involved in DNAreplication, said protein comprising an amino acid sequence as set forthin SEQ ID NO:2. In one embodiment, the nucleic acid sequence can be asset forth in SEQ ID NO:1.

In accordance with the present invention, there is also provided a DNAbinding protein involved in DNA replication. The protein comprises anamino acid sequence as set forth in SEQ ID NO:2.

In various embodiments, the DNA binding protein described above can beused in a screening method for identifying a compound binding to saidamino acid sequence, or in a screening method for identifying a compoundmodulating the binding of said DNA binding protein to a nucleic acidsequence. The compound preferably screened are those that inhibitcellular proliferation, such as cellular proliferation caused by cancer.Alternatively, the compound that can be screened are those that increasecellular proliferation.

Still in accordance with the present invention, there is also providedan antibody, a derivative or fragment thereof, binding to the DNAbinding protein described above and preventing said DNA binding proteinfrom binding to a nucleic acid sequence. The antibody, derivative orfragment thereof, can thus be used for preventing or decreasing cellularproliferation.

Further in accordance with the present invention, there is also provideda gene therapy comprising the step of introducing into a cell anexpression vector comprising a nucleic acid sequence encoding a DNAbinding protein involved in DNA replication, said protein comprising anamino acid sequence as set forth in SEQ ID NO:2. In one embodiment, thenucleic acid sequence can be as set forth in SEQ ID NO:1, wherein saidnucleic acid sequence in said expression vector once introduced in thecell encodes a protein with a DNA binding activity. Preferably, theprotein with a DNA binding activity has a sequence as set forth in SEQID NO:2.

Also in accordance with the present invention, there is provided amethod for screening compounds capable of modulating amino acids-DNAinteraction, said method comprising the steps of:

a) contacting in a medium the DNA binding protein described above the aDNA, said DNA binding protein being detectable and binding to said DNA;

b) adding to said medium) a compound to be screened for its capacity tomodulate the binding of amino acids to said DNA; and

c) detecting the effect on binding of the DNA binding protein to the DNAby the compound to be screened.

The present invention also provides a method for screening compoundscapable of modulating DNA replication, said method comprising the stepsof:

a) contacting in a medium a compound to be screened with a DNA bindingprotein comprising an amino acid sequence as set forth in SEQ ID NO:2;and

b) determining binding of said compound to the DNA binding protein,wherein detection of binding is indicative that said compound is capableof modulating DNA replication.

The present invention further provides a method for screening compoundscapable of modulating DNA replication, said method comprising the stepsof:

a) contacting in a medium a compound to be screened with a DNA bindingprotein comprising an amino acid sequence as set forth in SEQ ID NO:2;and

b) determining binding of said compound to the DNA binding protein,wherein detection of binding is indicative that said compound is capableof modulating DNA replication.

In accordance with the present invention, there is also provided amethod for screening compounds capable of modulating cell proliferation,said method comprising the steps of:

a) contacting in a medium a compound to be screened with a DNA bindingprotein comprising an amino acid sequence as set forth in SEQ ID NO:2;and

b) determining binding of said compound to the DNA binding protein,wherein detection of binding is indicative that said compound is capableof modulating cell proliferation.

In accordance with the present invention, there is additionally provideda method for screening compounds capable of modulating proliferation,said method comprising the steps of:

a) contacting in a medium a compound to be screened with a DNA bindingprotein comprising an amino acid sequence as set forth in SEQ ID NO:2;

b) adding DNA to said medium; and

c) determining binding of said compound to the DNA binding protein,wherein detection of the binding is indicative that said compound iscapable of modulating cell proliferation.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description will be more fully understood in viewof the drawings in which:

FIG. 1 is a photograph showing the Yeast One-Hybrid Assay;

FIG. 2 shows the target sequence effects on DUE-B binding;

FIG. 3 is an illustration of the DUE-B cDNA (SEQ ID NO:1) and protein(SEQ ID NO:2) sequences;

FIGS. 4A and 4B show the degree of homology between DUE-B from humansand other organisms, in the form of an evolutionary tree (FIG. 4A) andpercent identity indicated on the chart at the bottom (FIG. 4B);

FIGS. 5A and 5B are photographs showing mRNA and protein expression;

FIGS. 6A to 6L illustrate photographs showing immunocytochemistry ofHeLa cells expressing 6his, V5 epitope-tagged DUE-B, visualized in thecell nuclei by phase contrast microscopy (FIGS. 6A to 6D), Hoechststaining (FIGS. 6E to 6H), and FITC-conjugated V5 monoclonal antibody(FIGS. 6I to 6L);

FIG. 7 illustrates photographs showing the distribution of DUE-B in thenuclei and the supernatant;

FIG. 8 is a plot diagram of Sephacryl™ S-200 HR gel filtration ofrecombinant DUE-B;

FIG. 9 is a photograph showing the results of HeLa whole cell extractchromatographed on Sephacryl™ S-200 HR as in FIG. 8;

FIGS. 10A and 10B are photographs showing in vitro phosphorylation;

FIG. 11 is a plot diagram of ATPase chromatography;

FIG. 12 is a graph of the quantification of ATPase activity present inDUE-B;

FIG. 13 is a photograph showing that DUE-B and Replication Protein A(RPA) cooperate to affect DNA topology;

FIG. 14 illustrates an electrophoretic mobility shift analysis (EMSA);

FIG. 15 shows electrophoretic mobility shift assays using DUE-B and HeLanuclear extract bound to a DUE/ARS probe in the presence of poly-dI-dCand the indicated competitors;

FIGS. 16A and 16B are photographs showing chromatography of DUE-B inXenopus oocyte extract;

FIG. 17 is a photograph showing the results of chromatography ofrecombinant (baculovirus expressed) DUE-B added to HeLa cell nuclearextract;

FIGS. 18A to 18D illustrate replication in Xenopus oocyte extracts;

FIGS. 19A and 19B are photographs showing that baculovirus expressedDUE-B inhibits sperm chromatin replication;

FIG. 19C is a graph showing the effect of 30′ preincubation of extractswith control Sf9 lysate or recombinant DUE-B;

FIGS. 20A to 20D illustrate the interaction of DUE-B with chromatin;

FIG. 21 demonstrates that DUE-B antibody co-immunoprecipitates a 180 kDaprotein from Xenopus extracts;

FIG. 22A shows typical results of chromatography experiments usingsteroids passed through the column in the absence of DUE-B (hashedlines) and in the presence of DUE-B (solid lines);

FIG. 22B shows the retention times measured for several differentsteroids;

FIGS. 23A to 23C illustrate the development of the DUE-B assay designedin the present invention; and

FIGS. 24A and 24B show a test screen using selected steroids to measuretheir effects on the binding of DUE-B to dsDNA.

The embodiments set forth in the drawings are illustrative in nature andare not intended to be limiting of the invention defined by the claims.Moreover, individual features of the drawings and the invention will bemore fully apparent and understood in view of the detailed description.

DETAILED DESCRIPTION

In the figures and throughout the application, references are made tospecific compounds by a numeral reference. Table 1 below provides thecorrespondence between the numeral reference and the actual compounds.

TABLE 1 LIST OF COMPOUNDS USED AND/OR TESTED Steroid number (fromSteraloids) Steroid name [TRIVIAL (IUPAC)] P650 1-DEHYDROCORTISOL,PREDNISOLONE (1,4-pregnadien-11b,17,21-triol-3,20-dione) E87017a-ESTRADIOL (1,3,5(10)-estratriene-3,17a-diol) E950 17b-ESTRADIOL[1,3,5(10)-estrien-3,17-diol-17-acetate] E952 17b-ESTRADIOL 17-ACETATE[1,3,5(10)-estratriene-3,17b-diol] E1130 2-HYDROXYESTRONE[1,3,5(10)-estratrien-2,3-diol-17-one] E1140 2-METHOXYESTRONE 3-METHYLETHER [1,3,5(10)-estratrien-2,3-diol-17-one 2,3-dimethyl ether] E125016a-HYDROXYESTRONE [1,3,5(10)-estratrien-3,16a-diol-17-one] E140016-KETO-17b-ESTRADIOL [1,3,5(10)-estratrien-3,17b-diol-16-one] E2300ESTRONE [1,3,5(10)-estratrien-3-ol-17-one] E2301 ESTRONE ACETATE[1,3,5(10)-estratrien-3-ol-17-one acetate] E2470 2-HYDROXYESTRADIOL[1,3,5(10)-estratrien-2,3,17b-triol] E2500 4-HYDROXYESTRADIOL[1,3,5(10)-estratrien-3,4,17b-triol] E2600 ESTRIOL[1,3,5(10)-estratriene-3,16a,17b-triol] E2602 ESTRIOL 16-ACETATE[1,3,5(10)-estratrien-3,16a,17b-triol 16-acetate] E2695 ESTRIOL3-HEMISUCCINATE [1,3,5(10)-estratrien-3,16a,17b-triol 3-hemisuccinate]E2696 ESTRIOL 16-HEMISUCCINATE [1,3,5(10)-estratrien-3,16a,17b-triol16-hemisuccinate] E2750 ESTRIOL TRIACETATE[1,3,5(10)-ESTRATRIEN-3,16α,17β-TRIOL TRIACETATE] E2800 16,17-EPIESTRIOL[1,3,5(10)-estratrien-3,16,17-triol] E2850 16-EPIESTRIOL[1,3,5(10)-estratrien-3,16,17-triol] Q2600 PROGESTERONE[4-pregnene-3,20-dione] Q3880 HYDROCORTISONE(4-pregnen-11b,17,21-triol-3,20-dione) A6950 TESTOSTERONE[4-androsten-17b-ol-3-one]

Replication of the genome is essential for cell division and isregulated so that the entire DNA is duplicated once during the cellcycle. To accomplish the exact duplication of the genome, eukaryoticcells regulate where replication initiates along the chromosome, andwhen replication begins during the cell division cycle. Mutations inthese controls can lead to chromosome instability, cancer, or cell death(Hartwell, L. H., and M. B. Kastan, Science 266:1821-8, 1994).

The sites where the initiation of replication is controlled are termedreplication origins. In S. cerevisiae, chromosomal replication originscloned in plasmids display autonomous replicating sequence (ARS)activity, and characteristically comprise a set of modular elementsincluding an ARS consensus sequence (ACS) binding site for the yeastinitiator protein, the origin recognition complex (ORC) (Newlon, C. S.,and J. F. Theis, Curr. Opin. Genet. Dev. 3:752-758, 1993). Other origincomponents include a region of helical instability termed a DNAunwinding element (DUE), and transcription factor binding sites that maypromote the assembly of replication complexes through protein-proteininteractions and modulations of chromatin structure. In mammalian nucleino consensus DNA sequence has been identified that is analogous to theyeast initiator protein binding site (Cimbora, D. M., and M. Groudine,Cell 104:643-646, 2001). Instead the feature most common to mammalianorigins is not a DNA sequence but a structure, the DNA unwindingelement, DUE (Dobbs, D. L., et al., Nucleic Acids Res. 22:2479-2489,1994).

A replication origin in the 5′ flanking region of the human c-myc genewas first identified in the laboratory of one of the inventors (Leffak,M., and C. D. James, Mol. Cell. Biol. 9:586-593, 1989; McWhinney, C.,and M. Leffak, Nucleic Acids Res. 18:1233-1242, 1990; and McWhinney, C.,and M. Leffak, 1988. Episomal persistence of a plasmid containing humanc-myc DNA, vol. 6. CSH Laboratory Press, New York). Subsequently thisconclusion was confirmed by Vassilev and Johnson by PCR mapping of DNAnascent strands, and by others (Phi-van, L., et al., J. Biol. Chem.273:18300-18307, 1998; Rein, T., et al., J. Biol. Chem. 274:25792-80025,1999; Tao, L., et al., J. Cell. Biochem. 78:442-457, 2000; Tao, L., etal., J. Mol. Biol. 273:509-518, 1197; and Vassilev, L., and E. M.Johnson, Mol. Cell. Biol. 10:4899-4904, 1990). The inventors have alsoshown that the c-myc 2.4 kb HindIII/XhoI fragment endows the plasmidpNeo.Myc-2.4 with autonomously replicating sequence (ARS) activity whentransfected into HeLa cells, and in human cell free extracts in vitro(Berberich, S., A., et al., J. Mol. Biol. 245:92-109, 1995; Malott, M.,and M. Leffak, Mol. Cell. Biol. 19:5685-5695, 1999; McWhinney, C., andM. Leffak, Nucleic Acids Res. 18:1233-1242, 1990; McWhinney, C., and M.Leffak, 1988, Episomal persistence of a plasmid containing human c-mycDNA, vol. 6. CSH Laboratory Press, New York; McWhinney, C., et al., DNACell Biol. 14:565-579, 1995; Trivedi, A., et al., DNA Cell Biol.17:885-896, 1998). Replication in vitro initiates in the c-myc insert ofthe plasmid as demonstrated by two dimensional electrophoresis, electronmicroscopy and nascent strand polarity mapping and closely correspondsto the c-myc initiation zone deduced by Ishimi et al. (Ishimi, Y., etal., Mol. Cell. Biol. 14:6489-6496, 1994).

Computer analysis of the nucleotide sequence in the 5′ flanking DNA ofthe human c-myc gene predicted several regions of inherently bent orrigid DNA. These predictions were confirmed by two dimensional gelelectrophoresis of c-myc restriction fragments. Nuclease digestionrevealed a series of positioned nucleosomes and nuclease hypersensitivesites in the 2.4 kb region upstream of the c-myc promoters (Kumar, S.,and M. Leffak, J. Mol. Biol. 222:45-57, 1991; and Kumar, S., and M.Leffak, Nucleic Acids Res. 17:2819-2833, 1989).

This unique chromatin arrangement was stable when the 2.4 kbHindIII/XhoI fragment containing the c-myc ARS element was translocatedto other regions of the HeLa genome in an adeno-associated virus/c-mycconstruct, indicating that the structure is established by the bendingor other sequence-directed features of the DNA. Several transcriptionfactor consensus binding sites are present in the 5′ flanking region ofthe human c-myc gene (Michelotti, G. A., et al., Mol. Cell. Biol.16:2656-2669, 1995). One of these, a CTF/NF1 binding site, is close toDNase hypersensitive site II1 and a predicted DUE whose calculated freeenergy cost of unwinding is comparable to those of functional ARSelements in S. cerevisiae. The DUE is inside a region of ˜100 bp thatcontains three 10/11 matches to the S. cerevisiae ARS consensussequence. Comparison of the amino acid expressed by DUE with sequenceson NCBI resulted in an almost perfect match with a predicted proteinfrom a Homo sapiens histidyl-tRNA synthetase mRNA posted by Mao et al.(AF332356). However, Moa et al. never suggested that the predictedprotein would have DNA binding properties. The DUE/ARS region spans thec-myc far upstream element (FUSE) which is KMnO₄ reactive in HeLa cellsand is likely stabilized in an unwound state by interaction with theFUSE-binding protein FBP (Bazar, L., et al., J. Biol. Chem.270:8241-8248, 1995; Duncan, R., et al., Genes Dev. 8:465-480, 1994;Michelotti, G. A., et al., Mol. Cell. Biol. 16:2656-2669, 1996).

Mapping of DNA nascent strands confirmed that replication initiates atmultiple sites within and flanking the 2.4 kb c-myc core origin at itsendogenous location. To test whether this region satisfies the geneticdefinition of a chromosomal replicator which is able to promote its ownreplication and that of flanking chromosomal sequences, the core c-mycorigin, or control DNA, was integrated at the same ectopic chromosomalsite in human cells using the S. cerevisiae FLP recombinase enzyme(Malott, M., and M. Leffak, Mol. Cell. Biol. 19:5685-5695, 1999). Theabundance of short nascent DNA strands at the chromosomal acceptor sitewas quantitated before and after targeted integration of the originfragment and showed that the c-myc origin DNA substantially increasedthe amount of nascent DNA relative to the level at the unoccupiedacceptor site, and when compared to the level of nascent strands afterinsertion of control DNA. These results provided biochemical and geneticevidence for the replicator activity of the 2.4 kb region of c-mycorigin DNA.

The same system was used to demonstrate that removal of the DUE/ARSregion decreased c-myc chromosomal origin activity by more than half.Thus, the DUE/ARS is an essential component of the mammalian c-mycorigin, as it is in yeast origins. To identify proteins that mightregulate origin activity by binding to the DUE, a yeast one hybrid assaywas used. The present application describes the identification andcharacterization of a novel HeLa protein, DUE-B, which bindsspecifically to the c-myc DUE in vivo.

Results

The DNA unwinding element and ARS flanking DNA (102 bp) of the humanc-myc gene was used as a bait sequence to isolate cognatesequence-specific or structure-specific binding proteins in a yeastone-hybrid screen. The DUE/ARS was cloned upstream of a His3 reportergene promoter and integrated into a his-S cerevisiae strain. Due to lowlevel, leaky expression of the bait construct stable integration of thereporter could be selected for on his-plates. The reporter yeast strainwas transfected with a HeLa cDNA library (>10⁶ cDNA cfu) cloned in thepGAD-GH vector, to produce fusion proteins containing the Gal4transcription activation domain and HeLa proteins. Transfected yeastwere selected for plasmid retention (leu+) and elevated expression ofthe His3 reporter in the presence of 3-aminotriazole. One cDNA, pGK16B,resulted in large colony growth under selective conditions (FIG. 1). InFIG. 1, reporter yeast containing a histidine reporter gene (HIS3)downstream of the wild type c-myc DUE/ARS element, wild type DUE/mutantARS element, or mutant DUE/mutant ARS element were transformed with thepGK16B plasmid (encoding the DUE-B protein) and grown on his-medium withthe histidine anti-metabolite 3-AT (5 mM). The presence of DUE-Bpromotes the expression of His3 reporter gene and yeast growth only incells containing the wild type DNA binding site for DUE-B. Similarrelative growth rates for the three types of transformants were observedon his-medium with 0, 5, 10, or 15 mM 3-AT. FIG. 2 illustrates thegrowth rates in liquid culture (+5 mM 3-AT) of the yeast reporterstrains containing the wild type c-myc DUE/ARS element (DWAW), wild typeDUE/mutant ARS element (DWAM), mutant DUE/wild type ARS element (DMAW)or mutant DUE/mutant ARS element (DMAM). It is to be noted that mutationof the ARS consensus site for ORC binding enhances the effect of DUE-Bbinding (increased growth rate), while mutation of the DUE slows growth.This data shows that the unwinding element is critical for theactivation of the c-myc ARS.

Isolation of pGK16B and retransformation into the original reporterstrain (DUE/ARS WT) resulted in robust colony growth. Transformation ofpGK16B into otherwise isogenic yeast containing point substitutions inthe ARS Sequences slightly suppressed growth, while pGK16B could notsustain growth of otherwise isogenic yeast containing substitutions inthe ARS and DUE of the reporter. The protein encoded by pGK16B thereforebinds to the wild type DUE/ARS and enables the Gal4 transcriptionactivator to activate expression of the His3 reporter. In contrast,mutation of the ARS region of the DUE/ARS bait decreased reporterexpression, suggesting that the endogenous yeast replication initiatorcomplex ORC may interact with DUE-B during DNA binding. Mutation of theDUE and ARS elements eliminated reporter gene expression, indicatingthat the DUE region of the bait is essential for DUE-B binding. ThepGK16B cDNA was therefore renamed DUE-B to denote its affinity for DUEbinding in vivo.

Sequencing of the DUE-B cDNA revealed an open reading frame of 209 aminoacids (FIG. 3). DUE-B amino acids 29-147 are strongly (>90%) homologousto a domain of unknown function (DUF154) evolutionarily conserved inbacteria, yeast, and mammals. C-terminal to the DUF154 homology is acoiled-coiled domain characteristic of protein interaction sites,followed by a region of C-terminal homology (>50%) to segments of thehuman androgen receptor protein and to the ERK5 nuclear MAP kinase. TheDUE-B gene spans seven exons on chromosome 20, with the proposedinitiator methionine located in exon 2. In FIG. 3, the DUE-B cDNAsequence from the pGK16B plasmid is shown. Below is the one letter aminoacid translation of the sequence. A protein of 209 amino acids ispredicted. The putative initiator methionine ATG occurs in exon 2.Black/grey underlines indicate successive exons. The asterisk in exon 7indicates the putative stop codon.

As can be seen from FIGS. 4A and 4B, the degree of conservation betweenhuman and yeast sequences (45%) indicates the presence of some essentialregions of the protein.

Northern blot analysis revealed a single species of 1.35 kb DUE-B mRNA(FIG. 5A). FIG. 5A reports Northern blot analysis of HeLa RNA probedwith DUE-B cDNA. Lane 1 of FIG. 5A has been loaded withcross-hybridizing size marker RNA, whereas lanes 2 and 3, were loadedwith HeLa RNA.

DUE-B was cloned into the pTRC-His vector and expressed in E. coli. Theprotein was purified by nickel column chromatography and used to preparepolyclonal antibody. Western blot analyses showed that approximately 75%of DUE-B protein is found in the cytoplasmic fraction when cells arelysed in mild detergent (FIG. 5B). In FIG. 5B, proteins from nuclear orcytoplasmic extracts were separated by SDS-PAGE and western blots wereprobed with a polyclonal anti-DUE-B antibody. The nuclear fraction ofDUE-B can be extracted with moderate to high salt (0.5-1.0 M NaCl) andby DNase1 digestion, indicating that the nuclear fraction of DUE-B isbound to DNA. The intracellular distribution of DUE-B observed inasynchronous cells did not change when cells were arrested in mitosiswith nocodazole, or in S phase with aphidicolin or hydroxyurea. Theseresults were consistent with the observation that the level of DUE-BmRNA did not change appreciably over the course of the cell cycle.

To assess the distribution of DUE-B in intact cells an epitope tagged(V5, myc tags) version of the protein was expressed in HeLa cells.Immunocytochemical analysis using anti-myc antibody (FIGS. 6I to 6L) oranti-V5 antibody revealed that the expressed protein was localized tothe nucleus. In contrast, control reactions using the same antibodies tomonitor the distribution of a V5, his6-tagged MDM2 protein displayedonly the expected cytoplasmic fluorescence. These observations show thatDUE-B is located in the nucleus in intact HeLa cells.

In FIG. 7, it is shown that DUE-B is released into the supernatant (S)from pelleted (P) HeLa nuclei by micrococcal nuclease digestion (lanes5, 6) or HaeIII restriction DNase digestion (7, 8). A small amount ofDUE-B is released from nuclei in the presence of RNase (lanes 3, 4),similar to the amount of DUE-B released by incubation at 37° C. in theabsence of exogenous nuclease (lanes 1, 2). Thus, DUE-B appears to bespecifically bound to chromatin in nuclei.

The presence of a coiled-coil domain in the protein implied that DUE-Bmight form homo- or heteromeric complexes. The elution profile of thebacterial expressed DUE-B protein on Sephacryl™ S-200 columnchromatography was consistent with its monomeric molecular weight of 26kDa (FIG. 8). In FIG. 8, a Sephacryl™ S-200 column was calibrated usingcatalase, aldolase, BSA, chymotrypsinogen and cytochrome C. Acalibration curve was generated to determine the molecular weight ofDUE-B recombinant proteins (c-myc, 6his epitope tagged) produced in E.coli and SF9 insect cells. The elution peaks of the recombinant proteinswere determined by ELISA. A monoclonal antibody against the c-mycepitope was used to assay the rGK16B produced in E. coli. Polyclonalrabbit antibody against DUE-B expressed in E. coli was used to assay theDUE-B produced in insect cells. One major peak was observed in eachELISA. The E. coli generated protein has an apparent molecular weight of26.4 kDa (monomeric) while the insect expressed protein has an apparentmolecular weight of 54.7 kDa. In contrast, when DUE-B was purified frominsect cells infected with recombinant baculovirus, the recombinantDUE-B eluted as a dimer, near 50 kDa. Thus, expression in eukaryoticcells may enhance DUE-B protein-protein interaction by alteration inprotein folding or other posttranslational modifications. To determinewhether DUE-B existed in monomeric or multimeric states in HeLa cells,cell extracts were chromatographed. As seen in FIG. 9, endogenous DUE-Beluted at a molecular weight corresponding to ca. 50-54 kDa, with asmall percentage of the protein eluting near the void volume (fraction87; 250 kDa). In FIG. 9, fractions were separated by SDS-PAGE andvisualized by western blotting with the anti-DUE-B antibody.

A crude preparation of his-tagged DUE-B expressed in a baculovirusvector and purified on a nickel affinity column displayed ATPaseactivity and the ability to be phosphorylated in vitro withgamma-³²P-ATP in the absence (lane 1) or presence (lane 2) of histones(FIG. 10A). Similarly, DUE-B immunoprecipitated with preimmune serum(lane 3) or DUE-B antiserum (lane 4) from HeLa cell extracts alsocopurified with kinase activity and could be phosphorylated withgamma-³²P-ATP (FIG. 10B). However, immunoprecipitation of cell extractslabeled in vivo with ³²P-phosphoric acid did not reveal a significantlevel of DUE-B phosphorylation. Thus, DUE-B may be transientlyphosphorylated or unphosphorylated in vivo.

Column chromatography revealed that the kinase activity associated withthe baculovirus expressed DUE-B was not an inherent activity in theprotein, fractionating from the immunoreactive DUE-B in early and lateeluting peaks (FIG. 8). In FIG. 11, DUE-B expressed in baculovirusinfected insect cells was chromotographed as in FIG. 8. To locate theeluted DUE-B, fractions were assayed by ELISA using DUE-B antibody or6his antibody. ATPase activity was monitored by thin layerchromatography of alternate fractions incubated with gamma-³²P-ATP.However, the ATPase activity co-eluted with the DUE-B immunoreactivematerial, indicating that the DUE-B protein possesses intrinsic ATPaseactivity. Quantitation of the DUE-B ATPase activity by thin layerchromatography showed that approximately 30 fmol gamma-³²P-ATP werehydrolyzed per hour per fmol of DUE-B (FIG. 12). In FIG. 12, timecourses of ATPase activity were measured for three concentrations ofDUE-B protein.

On a chloroquine agarose gel of plasmid DNA, changes in the distributionof bands a, b, c, d in FIG. 13 indicate that DUE-B plus RPA increasesplasmid supercoiling by 1-2 turns more than either protein alone(compare lanes 3, 4, 5) and this effect is potentiated by ATP (comparelanes 8, 10, 11 with 3, 4, 5).

An electrophoretic mobility shift assay (EMSA) was used to test whetherthe baculovirus expressed DUE-B protein could bind to DNA in vitro. Whenadded to a radiolabeled c-myc DUE probe in the absence of nonspecificcompetitor (poly dI-dC), DUE-B produced a strongly retarded protein-DNAcomplex band (FIG. 14, lanes 1-4). Increasing the concentration of polydI-dC (lanes 5-12) reduced the levels of DUE-B bound DNA, suggestingthat DUE-B can bind DNA nonspecifically. When HeLa cell cytoplasmicextract was added to the c-myc DUE probe in the absence of poly dI-dC anintense band was observed that was not affected by the addition of DUE-B(lanes 13-16). With the addition of the nonspecific competitor polydI-dC, however, a novel band appeared that was dependent on the additionof DUE-B (asterisks, lanes 17-24). These results indicate that DUE-Binteracts with proteins released in the HeLa cytosol to form a specificcomplex on DNA. Similar to the results obtained when cytosol was addedto the c-myc DUE probe, when nuclear extract from HeLa cells was addedto the c-myc DUE probe in the absence of poly dI-dC an intense band wasobserved that was not affected by the addition of DUE-B (lanes 25-28).In the presence of competitor for nonspecific binding, however, a novelband appeared that was dependent on the addition of DUE-B (brackets,lanes 32 and 36). These results indicate that DUE-B interacts with HeLanuclear proteins to form specific complexes on the c-myc origin DNA. InFIG. 14, a 123 bp fragment of the c-myc replication origin containingthe DUE/ARS region was labeled with alpha-³²P-dCTP by PCR. 25 fmol weremixed with recombinant DUE-B purified by Ni-NTA affinity chromatographyfrom SF9 insect cells. EMSAs were performed in the presence of aninhibitor of non-specific DNA binding, poly dI-dC, HeLa cytoplasmicextract or HeLa nuclear extract as indicated. The labeled origin DNA wasvisualized by autoradiography. The data show that purified DUE-B proteinalters the electrophoretic migration pattern of origin DNA bound by HeLaproteins.

In FIG. 15, competition EMSA reveals the sequence-selective binding ofDUE-B: protein complexes. DUE-B changes the binding pattern of HeLanuclear extract proteins (compare lanes 3, 6 with 5, 7 and 8). Note alsothe competition for DUE-B: protein complex binding to the probe by DWAWbut not mutant DUE/ARS sequences DMAW, DWAM, DMAM.

As shown during the chromatography of HeLa extracts, virtually all ofthe endogenous DUE-B protein migrates as a dimer of ca. 50 kDa duringgel exclusion, although a minute amount of the protein can reproduciblybe detected at a substantially higher molecular weight (FIG. 9, fraction87, arrow). Since it can be estimated that fewer than 10% ofasynchronously dividing HeLa cells are in that portion of the cell cyclewhen pre-replicative complexes are assembled we sought for DUE-Binteracting proteins in the Xenopus oocyte extract system, which ispoised for rapid and efficient DNA replication. Baculovirus expressedrecombinant DUE-B protein was added to a Xenopus oocyte high speedcytosol extract in the presence of protease inhibitors, and the mixturechromatographed on Sephacryl™ S-200. As shown in FIG. 16A, the exogenousrDUE-B eluted as a high molecular weight (>250 kDa) complex which couldbe detected with anti-DUE-B antibody but not preimmune serum (FIG. 16B).The same result was obtained whether the extract had been treated withRNase (FIGS. 16A and 16B) or not suggesting that the exogenous DUE-B wasrapidly modified in the Xenopus extract or complexed with Xenopusproteins. In FIGS. 16A and 16B, DUE-B was mixed with Xenopus oocyteextract and the mixture chromatographed as in FIG. 8. Duplicate aliquotswere separated by SDS-PAGE gels and Western blotted with preimmuneantiserum or DUE-B antiserum. The profiles show that the exogenous DUE-Belutes with a molecular size of ca. 250 kDa while the putativeendogenous crossreactive DUE-B (ca. 26 kDa) elutes as a ca. 54 kDadimmer.

Note in FIG. 17 that a major portion of the recombinant DUE-B and asmall, but reproducible amount of the endogenous DUE-B (HDUE-B) elutewith a high molecular weight near the void volume of the column.

The anti-DUE-B antibody also detected a second band in the Xenopuscytosol preparation that eluted at an approximate molecular weight of 50kDa. Based on its immunoreactivity with anti-DUE-B antibody but notpreimmune serum, its molecular weight on SDS-PAGE (24 kDa) and itschromatographic elution as a ˜50 kDa dimer, we speculate that this mayrepresent the endogenous Xenopus DUE-B protein.

To test the effect of DUE-B on DNA replication, high molecular weight(phage lambda) DNA was preincubated in the Xenopus oocyte high speedextract. DUE-B was subsequently added with alpha-³²P-dCTP and an aliquotof the oocyte membrane fraction to yield a replication competentextract. A time course showed that DUE-B transiently inhibited DNAreplication (FIG. 18A). In FIG. 18A, plasmid pNeo.Myc-2.4 DNA wasincubated in oocyte extract for one hour prior to the addition ofalpha-³²P-dCTP (=time zero) with or without the addition of DUE-B. WhenDUE-B was preincubated with the extract and DNA before the addition ofalpha-³²P-dCTP and replication was assayed at 20 minutes, preincubationwith DUE-B was seen to inhibit replication further (FIG. 18B). In FIG.18B, phage lambda DNA was preincubated with Xenopus oocyte extract forone hour with or without DUE-B, prior to the addition of alpha-³²P-dCTP.Replication was measured 20 minutes after the addition ofalpha-³²P-dCTP. A possible explanation for the transient inhibition ofDNA replication by DUE-B is that the protein succumbs to degradationduring incubation in the Xenopus extract. To test this directly, DUE-Bprotein levels were determined by Western blotting following addition ofbaculovirus expressed protein to the Xenopus extract. However, as seenin FIG. 18C, the exogenous DUE-B is stable in the extract, againsuggesting that the exogenous DUE-B is rapidly modified when added tothe Xenopus extract. In FIG. 18C, baculovirus expressed DUE-B (1 μg) wasadded to Xenopus extract and aliquots were removed at the indicatedtimes for SDS-PAGE and Western blot analysis. The relative amount ofDUE-B remaining at each time point has been corrected for the amount ofXenopus cross-reacting band co-migrating with the baculovirus DUE-B. Todetermine whether DUE-B has an effect on replication through analteration in template structure, plasmid DNA was added to the Xenopusextract in the presence or absence of exogenous DUE-B. The addition ofDUE-B increased the amount of supercoiled (form I) plasmid relative tonicked form II plasmid. Thus, one effect of DUE-B may be to increase DNAsuperhelicity in the extract, possibly by nucleosome loading (FIG. 18D).In FIG. 18D, plasmid DNA was added to the Xenopus extract in the absenceor presence of DUE-B and analyzed by agarose gel electrophoresis.Addition of DUE-B increased the relative amount of supercoiled plasmidDNA.

In FIGS. 19A to 19C, sperm chromatin and alpha-³²P-dCTP were added toXenopus oocyte extracts in the presence or absence of DUE-B. The DNA waspurified, electrophoresed on agarose gels, and the incorporatedradiolabel quantitated. DUE-B expressed in baculovirus infected Sf9cells was more effective at inhibiting replication of the natural oocytesubstrate, sperm chromatin, when preincubated for 30′ with the extractthan when added simultaneously with the sperm chromatin.

In FIG. 20A, an ELISA assay shows that Xenopus oocyte extract reduces,but does not eliminate, saturable DUE-B binding to sperm chromatin. InFIG. 20B, oocyte extracts were centrifuged after the addition of spermchromatin in the presence or absence of DUE-B. Pellets wereimmunoblotted with anti-MCM7 antibody. As can be seen from FIG. 20C,DUE-B does not inhibit MCM7 loading (prereplication complex [pre-RC]formation) on chromatin. In FIG. 20D, it can be seen that DUE-B does notinhibit replication of single stranded DNA in oocyte extract. These datasuggest that DUE-B selectively inhibits replication of double strandedDNA, at a step after pre-RC formation.

FIG. 21 shows the results of immunodepletion of Xenopus oocytereplication extracts with anti-DUE-B antibody or preimmune serum. Notethat the anti-human DUE-B antibody not only removes Xenopus DUE-B fromthe extract but selectively precipitates a 180 kDa protein notprecipitated by the preimmune serum.

Steroid Binding of DUE-B

Steroid binding properties of DUE-B were assessed using purified 6his-V5tagged DUE-B immobilized to a Nickel-Sepharose™ column (via the 6histag) (FIGS. 22A and 22B). Steroids were passed through the column (50 ulof 100 nM) in the presence of 10 mM NH₄OAc (pH 7.4) and 10% MeOH. Flowthrough was analyzed for steroid content by mass spectroscopy. Due tothe fact that some steroids content by mass spectroscopy. Due to thefact that some steroids give a stronger signal by MS, signal strengthshave been normalized to show the effects of DUE-B on steroid retention.Note that in the presence of DUE-B, some selected steroids have a muchlonger retention time in the column. This indicates a strongerassociation of these particular steroids with DUE-B. In FIG. 22B, notethat marked increases in retention times are only seen with a subset ofsteroids, indicating that not only does DUE-B bind steroids, but it alsodisplays selectively in the steroids it binds.

A Novel High Throughput Assay for Measuring DUE-B DNA Binding

Described herein is the first generation of a novel assay fordetermining the effects of molecules on the association of DUE-B withdouble stranded DNA (dsDNA). This assay uses fluorescence polarizationto measure the interaction of DUE-B with dsDNA. This assay consists of 4components:

-   -   a) a dsDNA derived from the DUE sequence:

(SEQ ID NO: 3) (5′-GGAATATACA TTATATATTA AATATAGATC-3′)

-   -   Both the sense and antisense strands are labeled with FITC at        the 3′ end using a 6 carbon spacer.    -   b) Purified DUE-B. Human DUE-B tagged at the amino terminus with        the 6his and V5 tags is synthesized in baculovirus and purified        using Ni²⁺-Sepharose™ chromatography.    -   c) Reaction buffer (1×): 100 mN Tris HCl (pH 7.5), 800 mM NaCl,        10 mM EDTA, 100 mM β-mercaptoethanol, 1% (w/v) Tween-20™.    -   d) Fluorescence polarization plate reader (Tecan Polarion™ or        other equivalent device)

Methodology

A mastermix is prepared consisting of 1× reaction buffer, 2 nM labeledoligo and 0.125 or 0.250 ug DUE-B per 100 ul of mastermix. Compounds (inthis case steroids) are added to the bottom of a 96 well plate. 100 ulof mastermix is added to each well and allowed to equilibrate for 30sec. Fluorescence polarization is then measured.

In FIG. 23A, the effect of increasing concentrations of DUE-B on thepolarization of dsDNA was assessed. As expected, polarization of thedsDNA becomes saturated as the DUE-B protein concentration increases.Note that at 0.125 and 0.250 μg of DUE-B a reasonable shift inpolarization of dsDNA is seen. Using these set conditions (0.250 μgDUE-B), the effect of adding a non-specific DNA inhibitor (pdIdC) to thereaction (FIG. 23B) was tested. The loss of dsDNA polarization in thepresence of pdIdC indicates a reversible DNA association with DUE-B.FIG. 23C shows the temporal stability of these signals. It is observedthat the polarization was stable for at least 2 hours, making this assayvery feasible for high throughput screening.

FIG. 24A shows the extent of polarization of dsDNA by DUE-B in thepresence of different steroids at 10 uM. Note that E2850 gave a 100%increase in polarization, indicating a strong stimulatory effect on thedsDNA binding of DUE-B. FIG. 24B shows the validation of the results inFIG. 24A by retesting the compounds in two different concentrations ofDUE-B. Here the stimulatory effect of E2850 was duplicated.

Discussion

In accordance with the present invention, a novel protein, DUE-B, hasbeen isolated based on its selective affinity for the DUE/ARS sequenceof the human c-myc replication origin in a yeast one-hybrid assay.Inasmuch as the c-myc DUE/ARS contains three matches to the yeast ARSconsensus sequence, DUE-B is capable of binding to the DUE/ARS throughdirect association with DNA, through interaction with yeast originbinding proteins, or through a combination of forces. Interaction withorigin DNA through secondary interactions with yeast proteins issuggested by the reduction in reporter expression when the ARS consensuselements of the DUE/ARS binding site were mutated. Sequencing andtranslation of the DUE-B cDNA predicts a protein of 209 amino acids,with a molecular weight of ca. 26 kDa. Northern blot analysis revealed a1.35 kb mRNA, sufficient to encode a protein of 26 kDa and Westernanalysis using antibody raised against recombinant DUE-B cloned inbacteria detected the predicted ˜26 kDa protein in HeLa cells. Screeningof the NCBI Genbank database showed that highly homologous proteins arepredicted to occur in bacteria, yeast, and mice. DUE-B mRNA and proteinappear to be present at roughly constant levels throughout the cellcycle and agents that inhibit replication and elicit DNA damage responsepathways (e.g. aphidicolin, hydroxyurea) did not affect the levels ofDUE-B protein.

In the one-hybrid assay a nuclear localization signal becomes part ofthe HeLa library proteins expressed in the reporter yeast.Immunocytochemical analysis showed that DUE-B expressed in HeLa cellslocalized to the nucleus in the absence of an exogenous nuclearlocalization sequence. This observation was consistent with data showingthat a fraction of endogenous DUE-B could be recovered from nucleiisolated after HeLa cell lysis and released from the nuclei by salt orDNase extraction.

DUE-B cloned and expressed in bacteria chromatographed as a ˜26 kDamonomer on gel exclusion chromatography, while DUE-B cloned in abaculovirus vector and expressed in insect cells eluted as a ˜50 kDadimer, suggesting that expression in the eukaryotic insect cells mayresult in a different posttranslationally modified form of the protein,and that the posttranslational modification may influence DUE-Bfunction. Chromatography of HeLa extracts also revealed the ˜50 kDadimeric form of the endogenously synthesized DUE-B, along with a minoramount of DUE-B protein eluting at higher molecular weight (>250 kDa).When baculovirus expressed DUE-B was mixed with Xenopus oocyte extractsor HeLa extracts virtually all of the added DUE-B was found to elute asthe high molecular weight protein complex. In contrast, the putativeXenopus or HeLa DUE-B proteins eluted at the ˜50 kDa dimer position.These data suggest that the structure of the exogenous HeLa DUE-Bprotein may be modified in the Xenopus or HeLa extracts, which resultsin its association to a high molecular weight protein complex.

A prevalent modification of proteins involved in the assembly ofreplicative complexes is transient phosphorylation. Despite the presenceof seven casein kinase consensus target sites, radio labeling of HeLacells with ³²P-orthophosphate did not reveal in vivo phosphorylation ofimmunoprecipitated DUE-B. However, since immunoprecipitated HeLa DUE-Bor baculovirus expressed DUE-B could be phosphorylated in vitro bycopurifying kinases, it remains possible that DUE-B undergoes transientphosphorylation in vivo.

Proteins that bind and hydrolyze ATP are common in the initiation of DNAreplication. Consistent with the prediction of ATP and GTP bindingdomains in DUE-B, chromatography of the baculovirus expressed proteinshowed that the DUE-B dimer co-eluted with ATPase activity. Under thepresent assay conditions DUE-B hydrolyzed >0.5 fmol ATP per minute perfmol protein. For comparison, the calculated Vmax of purified yeast ORCis 0.27 fmol ATP hydrolyzed per min per fmol protein.

The binding of DUE-B to DNA in EMSA could be reversed by nonspecificcompetitor, suggesting that DUE-B possess a nonspecific affinity forDNA. Similar binding has been observed for the yeast ORC. However, inthe presence of cytoplasmic or nuclear extracts, DUE-B appeared to formheteromeric complexes that were resistant to nonspecific competition.The ability to form high molecular weight complexes was implied by thepresence of a small amount of early eluting DUE-B in HeLa extracts, andthe early elution of DUE-B added exogenously to Xenopus oocyte extracts.The data obtained in accordance with the present invention also suggestthat the anti-DUE-B antibody may have uncovered a crossreacting Xenopushomolog of DUE-B, and that these proteins may undergo distinctmodifications that affect their structure and function in the Xenopusextract. The association of DUE-B with heterologous proteins in solutionor bound to c-myc origin DNA suggests that methods for detectingprotein-protein interactions (yeast two-hybrid system, affinitychromatographic co-purification, co-immunoprecipitation) may revealnatural binding partners that interact with DUE-B to affect theinitiation of DNA replication.

Materials and Methods

Yeast One-Hybrid Assay

The wild type DUE/ARS region of the c-myc origin (nt 735-832; Genbankaccession number X00364) was cloned into the vector pHisi-1 andtransformed into S. cerevisiae strain YM4271 (MATa, ura3-52, his3-200,ade2-101, lys2-801, leu2-3, 112, trp1-901, tyr1-501, gal4-D512,gal80-D538, ade5::hisG) according to the manufacturer's directions. Thewild type and mutant DUE/ARS bait sequences are as follows:

Wild type: ATGAGAAGAA TGTTTTTTGT TTTTCATGCC GTGGAATAAC ACAAAATAAA(SEQ ID NO: 4) AAATCCCGAG GGAATATACA TTATATATTA AATATAGATC ATTTCAGG.ARS mutant: ATGAGAAGAA TGTTTTTTGC GCTTCATGCC GTGGAATAAC ACAGCGTAAA(SEQ ID NO: 5) AAATCCCGAG GGAATATACA TTATATATTT GTTATAGATC ATTTCAGG.DUE mutant/ARS mutant:ATGAGAAGAA TGTTTTTTGC GCTTCATGCC GTGGAATAAC ACAGCGTAAA (SEQ ID NO: 6)AAATCCCGAG GGAATGCACA TTGCATATTG CGCGTACGAT CATTTCAGG.

Transformants were selected for growth on his-medium (Clontech). Thereporter strain was transformed with a HeLa cDNA library cloned inpGAD-GH (Clontech Matchmaker) and colonies selected for growth at 30° C.on his-, leu-medium containing 15 mM 3-aminotriazole (3-AT). Plasmid wasisolated from crude yeast lysates and cloned in E. coli according tostandard procedures. DNA was sequenced on an Applied Biosystem 377 DNASequencer.

DUE-B Protein and mRNA Analysis

The cDNA insert of plasmid pGK16B encoding the DUE-B protein with aC-terminal his6 tag was cloned by PCR, inserted into the bacterialexpression vector pTRC-His and expression in E. coli was induced byIPTG. The protein was isolated on Ni-NTA columns (Qiagen) undernon-denaturing conditions following the manufacturer's instructions.Polyclonal antibody to DUE-B was produced commercially (Cocalico Corp)by injection of bacterial expressed DUE-B into rabbits. HeLa cells weresynchronized in S phase (1 μg/ml aphidicolin or 2 mM hydroxyurea,overnight), M phase (100 ng/ml or 400 ng/ml nocodazole, overnight). HeLacells were lysed using Popper buffers (Pierce) to yield nuclear andcytoplasmic fractions. Western blotting was performed on proteinsresolved on 12% SDS-PAGE gels transferred to Immobilon™ membranes bystandard procedures. For expression in insect cells using the MaxBac™kit (Invitrogen) DUE-B cDNA was cloned into the pBlueBac4.5 vector andcotransformed with Bac-N-Blue AcMNPV DNA into SF9 cells according to themanufacturer's directions.

Purified DUE-B (200-1000 ng) expressed in bacteria or insect cells, orcell extracts from HeLa cells (˜500 ng) or Xenopus oocytes (˜500 ng),were chromatographed on a one-meter Sephacryl™ S-200 column. Proteinelution was monitored by Western blot or by ELISA using antibodies toDUE-B or the his6 tag. ATPase activity was monitored by thin layerchromatography on PEI cellulose (Patrick, S. M., et al., Biochem.Biophys. Acta 1354:279-290, 1997). RNA was isolated using an RNAeasy kit(Qiagen) and DUE-B mRNA expression was monitored by Northern blotting oftotal RNA electrophoresed on denaturing formaldehyde/agarose gels usinga DUE-B cDNA probe labeled with alpha-³²P-dCTP by random primerextension.

Immunocytochemistry

DUE-B cDNA including myc and his6 epitope tags was subcloned into theeukaryotic expression vector pcDNA3.1 and transfected into HeLa cells.Forty-eight hours post-transfection the cells were fixed, permeabilized,and incubated with FITC conjugated antibody specific for the mycepitope. Cells were counterstained with Hoechst™ 33258 dye.

EMSA

A 123 bp fragment containing the c-myc DUE/ARS was labeled by PCR in thepresence of alpha-³²P-dCTP. The sequence of the probe is: GAAGGAATTCATGAGAAGAA TGTTTTTTGT TTTTCATGCC GTGGAATAAC ACAAAATAAA AAATCCCGAGGGAATATACA TTATATATTA AATATAGATC ATTTCAGGGA GCTCGAGAAA CAA (SEQ IDNO:7).

Recombinant DUE-B was obtained from SF9 insect cells and purified byNi-NTA affinity chromatography. Binding reactions were performed at 30°for 30 minutes and separated by 4% native PAGE at room temperature in0.5×TBE buffer prior to autoradiography.

Replication in Xenopus oocyte Extracts

Oocyte extracts were prepared according to published procedures and thereplication of plasmid pNeo.Myc-2.4 or phage lambda DNA monitored in thepresence of alpha-³²P-dCTP as described in Walter, J., and J. Newport(Walter, J., and J. Newport, Mol. Cell. 5:617-627, 2000) and in Walter,J., et al., (Water, J., et al., Mol. Cell. 1:519-529, 1998)

The specific illustrations and embodiments described herein areexemplary only in nature and are not intended to be limiting of theinvention defined by the claims. Further embodiments and examples willbe apparent to one of ordinary skill in the art in view of thisspecification and are within the scope of the claimed invention.

1. A method for screening compounds capable of modulating an interactionbetween a DNA binding protein comprising an amino acid sequence as setforth in SEQ ID NO: 2 and DNA comprising a c-myc replication origin,said method comprising the steps of: a) contacting in a medium a peptidecomprising an amino acid sequence as set forth in SEQ ID NO:2 and apolynucleotide comprising a sequence of a c-myc replication origin;wherein the peptide is detectably bindable to at least a portion of thec-myc replication origin polynucleotide sequence; b) adding to saidmedium a compound to be screened for its capacity to modulate thebinding of the peptide to the polynucleotide; c) determining binding ofsaid compound to said peptide; and d) detecting change in binding ofsaid peptide to said polynucleotide; wherein detection of binding ofsaid compound to said peptide and change in binding of said peptide tosaid polynucleotide is indicative that said compound is capable ofmodulating an interaction between a DNA binding protein comprising anamino acid sequence as set forth in SEQ ID NO: 2 and DNA comprising ac-myc replication origin.
 2. A method for screening compounds capable ofmodulating DNA replication, said method comprising the steps of: a)contacting in a medium a peptide comprising an amino acid sequence asset forth in SEQ ID NO:2 and DNA comprising a c-myc replication origin;wherein the peptide is detectably bindable to at least a portion of thec-myc replication origin; wherein binding of the peptide to the DNA isassociated with detectable DNA replication activity; b) adding to saidmedium a compound to be screened for its capacity to modulate DNAreplication; c) determining binding of said compound to said peptide;and d) determining change in DNA replication activity; wherein detectionof binding of said compound to said peptide and change in DNAreplication activity is indicative that said compound is capable ofmodulating DNA replication.
 3. A method according to claim 1 foridentifying a compound capable of inhibiting an interaction between aDNA binding protein comprising an amino acid sequence as set forth inSEQ ID NO: 2 and DNA comprising a c-myc replication origin, comprisingdetecting binding of the compound to be screened to said peptide andinhibition of binding of said peptide to said polynucleotide.
 4. Amethod according to claim 1, wherein the peptide is a polypeptide.
 5. Amethod according to claim 4, wherein the polypeptide is DNA UnwindingElement Binding Protein.
 6. A method according to claim 1, wherein thecompound to be screened is a steroid.
 7. A method according to claim 1,wherein the polynucleotide comprises a sequence of a c-myc replicationorigin DNA Unwinding Element, and the peptide is bindable thereto.
 8. Amethod according to claim 7, wherein the polynucleotide comprises asequence as set forth in SEQ ID NO:
 4. 9. A method according to claim 7,wherein the polynucleotide comprises a sequence as set forth in SEQ IDNO:
 3. 10. A method according to claim 2 for identifying a compoundcapable of inhibiting DNA replication, comprising detecting binding ofthe compound to be screened to said peptide and inhibition of DNAreplication activity.
 11. A method according to claim 2, wherein thepeptide is a polypeptide.
 12. A method according to claim 11, whereinthe polypeptide is DNA Unwinding Element Binding Protein.
 13. A methodaccording to claim 2, wherein the compound to be screened is a steroid.14. A method according to claim 2, wherein the DNA comprises a c-mycreplication origin DNA Unwinding Element, and the peptide is bindablethereto.
 15. A method according to claim 14, wherein the DNA comprises asequence as set forth in SEQ ID NO:
 4. 16. A method according to claim14, wherein the DNA comprises a sequence as set forth in SEQ ID NO: 3.17. A method for screening compounds capable of modulating aninteraction between a DNA binding protein comprising an amino acidsequence as set forth in SEQ ID NO: 2 and DNA comprising a c-mycreplication origin DNA Unwinding Element, said method comprising thesteps of: a) contacting in a medium a peptide comprising at least aportion of an amino acid sequence as set forth in SEQ ID NO:2 and DNAcomprising a c-myc replication origin DNA Unwinding Element; wherein thepeptide is detectably bindable to at least a portion of the c-mycreplication origin DNA Unwinding Element; wherein binding of the peptideto the DNA is associated with detectable DNA replication activity; b)adding to said medium a compound to be screened; c) determining bindingof said compound to said peptide; and d) detecting change in binding ofsaid peptide to said DNA; wherein detection of binding of said compoundto said peptide and change in binding of said peptide to said DNA isindicative that said compound is capable of modulating an interactionbetween a DNA binding protein comprising an amino acid sequence as setforth in SEQ ID NO: 2 and DNA comprising a c-myc replication origin DNAUnwinding Element.
 18. A method according to claim 17 for identifying acompound capable of inhibiting DNA replication, comprising: determiningchange in DNA replication activity; wherein detection of (i) binding ofsaid compound to said peptide, (ii) inhibition in binding of saidpeptide to said DNA, (iii) change in DNA replication activity, or (iv)combinations thereof, is indicative that said compound is capable ofinhibiting DNA replication.
 19. A method according to claim 18, whereinthe DNA comprises a c-myc replication origin DNA Unwinding Elementsequence as set forth in SEQ ID NO: 4.